Composition and Method for Modulating Plant Transformation

ABSTRACT

A plant culture medium composition for modulating plant transformation events, comprising a plant culture medium and an effective amount of at least one compound having a rare earth element component intermixed thereinto. The at least one rare earth element-containing compound is selected from the group comprising: CeCl 3 , LaCl 3 , and combinations thereof. Another embodiment relates to a method for modulating the frequency of plant transformation events. The method comprises the steps of providing the plant culture medium composition and contacting at least one plant with the plant culture medium composition. At least one cell from the at least one plant is transformed with a nucleic acid of interest. The presence of at least one transformation event is detected and quantified. The frequency of quantified transformation events is compared with a suitable control. Changes in quantified transformations events compared to the control are indicative of changes in the frequency of plant transformation events.

TECHNICAL FIELD

The present invention generally relates to plant growth media,particularly to plant growth medium compositions configured formodulating the frequency of plant transformation, and more particularlyto plant growth media compositions having a rare earth elementcomponent.

BACKGROUND ART

Agriculture is a multibillion-dollar industry that can be significantlyimpacted by even seemingly small improvements in methods or compositionsfor improving transfer of foreign genes into plants. Traditionally,methodologies based on sexual reproduction have been utilized for thetransfer of genes within plant species or between closely related plantspecies to improve crop qualities. The pace of crop improvement by suchmethodologies has been slow and limited, in part due to reliance onnaturally occurring gene variations in closely related species.

Advances in genetic engineering provide an alternative approach forintroducing foreign genetic information into plants, thereby resultingin transgenic plants that have acquired new beneficial characteristics.Genetic engineering of plants involves genetic transformation byintroducing foreign genetic material(s) in the form of a nucleic acidsuch as DNA, which encodes for one or more genes. Other transformationtechniques, which are all well known in this field, include somatichybridization by fusion of protoplasts and the induction of somaclonalvariations in order to induce genetic modifications.

The transfer of foreign genetic material into plants is commonlyperformed utilizing well-known gene transfer techniques such asAgrobacterium-mediated transformation. This technique utilizes strainsof Agrobacterium containing an engineered Ti plasmid to introduce thegenetic material of interest. Plant tissue is cut into small pieces andsoaked for about 10 minutes in an Agrobacterium suspension. Thesebacteria enable expression of the genetic material and producetransformants or transformed plants that exhibit profitable agronomiccharacteristics. Thus, it is possible to produce plants with certaindesirable characteristics such as resistance to herbicides, insects, andviral diseases.

Large economic expenses have been devoted to the development ofrecombinant DNA technology for manipulating genetic information inplants. For example, plant genes can be cloned, and desirable genes canbe recombined from unrelated organisms to confer new agriculturallyuseful traits to crops. Recombinant DNA technology has created a largergene pool available for crop improvement.

However, the benefit of these advances in bioengineering can only berealized if these genes of interest can be introduced into plantsreliably, consistently and economically. The increase in the efficiencyof transformation rates, even by as much as two-fold, can translate intosignificant cost savings with respect to expenditures such as technicalstaff salaries, material costs and energy costs.

There are a number of methods directed to improving plant transformationefficiencies. These methods are aimed at improving the health of thebacteria that is used for transformation, the health of recipienttransformed plants and the conditions during plant regeneration.

Plant transformation is by no means a routine matter. For manycommercially important crop plants, the efficiency or frequency oftransformation is calculated by dividing the number of transformedplants produced by each transformation attempt. Both the efficiency andfrequency is very low and highly variable among genetic lines andvarieties. Some highly desirable breeding lines exhibit extremely lowtransformation frequencies relative to other genetic lines of the samecrop species. In some cases, satisfactory levels of transformed plantcells and calli can be achieved from a transformation attempt, but suchtransformed cells and calli are resistant to regeneration intotransformed embryos and plants.

The prior art methods generally result in poor control over where andhow the DNA of interest is integrated into the plant genomic DNA duringtransformation. The introduced genetic material typically integratesrandomly and is mediated primarily via non-homologous end-joining thusleading to frequent inactivity of the transgene and/or modification ofthe genomic sequences due to integration of truncated copies of the DNA,multiple integrations, and deletions at the site of integration. Also,the prior art methods are only aimed at improving one of thetransformation steps in gene transfer.

It is known that double strand breaks are associated with transformationso that the foreign DNA of interest can integrate into the plant genomicDNA. Repair of the DNA strand breaks are mediated by two majormechanisms or pathways, namely non-homologous end-joining and homologousrecombination. Researchers have revealed that non-homologous end-joiningis an error-prone mechanism and frequently results in deletions and/orinsertions at the place of the repair where the integration hasoccurred. In contrast, homologous recombination is considered error-freeand, therefore, a more desirable mechanism for DNA integration duringplant transformation. However, non-homologous end-joining is thepredominant repair mechanism in plants. It has been shown that the ratioof non-homologous end-joining to homologous recombination is at leastabout 1000:1 in plants.

The inability to control where and how genes are integrated and theerrors introduced during transformation are major drawbacks of existingmethodologies in gene transformation. It is currently unclear whatfactors control the preferential utilization of non-homologousend-joining over homologous recombination for the repair of doublestrand breaks in plants. Both of these mechanisms play a role in theintegration of foreign DNA with respect to transformation. Given thatnon-homologous end-joining is the predominant mechanism utilized inplants, an increase in homologous recombination can lead to moreeffective integration of the desired gene, more intact “clean”integration and greater control in targeting genes to their desiredlocations.

SUMMARY OF THE INVENTION

Exemplary embodiments of the present invention are related to plantculture medium compositions and methods for modulating the frequency ofplant transformation events.

One exemplary embodiment of the present invention relates to a plantculture medium composition configured for modulating planttransformation events. The composition comprises a plant culture mediumand an effective amount of at least one compound having a rare earthelement component intermixed thereinto. According to one aspect, abaseline level for plant transformation events is provided by culturingat least one of a plurality of plant cells, at least one plant, andplant tissue on the plant culture medium.

In a further exemplary embodiment, on comparison to the baseline levelfor plant transformation events, the plant culture medium compositionincreases the number of plant transformation events. According to oneaspect, on comparison to the baseline level for plant transformationevents, the plant culture medium composition increases the number ofplant transformation events by at least one of 2-fold, 3-fold, 4-fold,5-fold, and 10-fold.

A further exemplary embodiment of the present invention relates to amethod for modulating the frequency of plant transformation events. Themethod comprises the steps of providing a plant culture mediumcomposition where the composition comprises a plant culture medium andan effective amount of at least one compound having a rare earth elementcomponent intermixed thereinto. At least one plant is then contactedwith the plant culture medium composition, and at least one cell fromthe at least one plant is transformed with a nucleic acid of interest.The presence of at least one transformation event is then detected andthe transformation events quantified. The frequency of the quantifiedtransformation events is then compared with a suitable control. Changesin the quantified transformations events compared to the control areindicative of a change in the frequency of plant transformation events.According to one aspect, changes in the quantified transformationscompared to the control, are an increase in the frequency of planttransformation events. According to another aspect, changes in thequantified transformations compared to the control are an increase inthe frequency of plant transformation events by at least one of 2-fold,3-fold, 4-fold, 5-fold, and 10-fold.

In another exemplary embodiment, the at least one rare earthelement-containing compound is selected from the group comprising:CeCl₃, LaCl₃ and combinations thereof. According to a one aspect, therare earth element containing compound is CeCl₃. According to anotheraspect, the rare earth element containing compound is LaCl₃. Accordingto a further aspect, the rare earth element containing compound is acombination of CeCl₃ and LaCl₃. According to yet another aspect, theCeCl₃, LaCl₃, and combinations thereof are each provided in an amountgreater than about 0.1 μM and less than about 3.0 μM.

In one exemplary embodiment, a suitable control is selected from thegroup comprising a stored dataset of results generated from studies ofthe presence and expression transformation events in one or morepopulation(s) of plants grown on the plant culture medium, a storeddataset of results generated from studies of the presence and expressionof transformation events in one or more population(s) of plant cellsgrown on the plant culture medium, a stored dataset of results generatedfrom studies of the presence and expression transformation events in oneor more population(s) of plant tissue grown on the plant culture mediumand combinations thereof.

Another exemplary embodiment of the present invention relates to amethod for transforming a plant cell. The method comprises the steps ofproviding a plant culture medium composition where the compositioncomprises a plant culture medium and an effective amount of at least onecompound having a rare earth element component intermixed thereinto. Aplurality of plant cells are contacted with the plant culture mediumcomposition and the plurality of plant cells are transformed with aselected nucleic acid. The presence of at least one transformation eventis detected and at least one transformed plant is regenerated from atleast one transformed plant cell.

Further aspects of the invention will become apparent from considerationof the ensuing description of preferred embodiments of the invention. Aperson skilled in the art will realize that other embodiments of theinvention are possible and that the details of the invention can bemodified in a number of respects, all without departing from theinventive concept. Thus, the following drawings, descriptions andexamples are to be regarded as illustrative in nature and notrestrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described, by way ofexample only, with reference to the attached Figures, wherein:

FIG. 1 is a block diagram showing the structure of a gene constructuseful for plant transformation with an exemplary embodiment of thepresent invention;

FIG. 2( a) is a block diagram showing homologous recombination eventswith the GUS marker gene, and FIG. 2( b) is a companion image showingplants transformed with the GUS-marker gene;

FIG. 3 is a chart showing the effects of different concentrations ofCeCl₃, LaCl₃, and a combination of CeCl₃, LaCl₃ on the homologousrecombination frequency in Nicotiana tabacum; and

FIG. 4 is a chart showing the effects of different concentrations ofCeCl₃ on the numbers of calli regenerated and the regeneration of stableN. Tabacum transformants.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention relate to methods and compositionsconfigured for modulating plant transformation, more particularly forincreasing plant transformation frequency. Some embodiments relate toplant culture medium compositions for modulating plant transformation,specifically homologous recombination. The present invention furtherrelates to methods for transforming plants and methods for modulatingthe frequencies of plant transformation. It was discovered by thepresent invention that growing plants on medium enriched with rare earthelements compounds, in particular cerium (III) chloride (CeCl₃) orlanthanum (III) chloride (LaCl₃), affects increases in the homologousrecombination rates of plants without causing physiological damage tothe plants.

According to IUPAC, rare earth elements or rare earth metals are acollection of seventeen chemical elements in the periodic table. Theprincipal sources of rare earth elements are the minerals bastnäsite,monazite, and loparite and the lateritic ion-adsorption clays.Well-known minerals that contain cerium and the light lanthanoidsinclude bastnaesite, monazite, allanite, loparite, ancylite, parisite,lanthanite, chevkinite, cerite, stillwellite, britholite, fluocerite,and cerianite. Lanthanum is found in some rare earth minerals, usuallyin combination with cerium and other rare earth elements. Cerium is themost abundant of the rare earth elements and cerium(III) compounds, forexample cerium(III) chloride, have uses as catalysts in organicsynthesis. Lanthanum has no known biological role however, it haspharmacological effects on several receptors and ion channels, and itsspecificity for the GABA receptor is unique among divalent cations.

Homologous recombination is a type of genetic recombination, a processof physical rearrangement occurring between two strands of DNA.Homologous recombination involves the alignment of similar sequences,formation of a Holliday junction, and breaking and repair, known asresolution, of the DNA to produce an exchange of material between thestrands. The process of homologous recombination naturally occurs inplants. Homologous recombination is the mechanism of crossing-over inmeiosis, and this mechanism creates diversity in the plant population.Breeders rely on this diversity when breeding new plant varieties. Thus,growing plants on a medium that is known to augment the rate ofhomologous recombination may also allow for higher diversity in theplant progeny grown on that same medium.

The present invention also relates to the addition of rare earthelements, particularly CeCl₃ and LaCl₃, in specific concentration rangesfor increasing the frequency of homologous recombination.

There are several technologies known in the art that may be used totransform plant cells with selected DNA molecules. These technologiesare well known to those persons skilled in the art and may include, butare not limited to: (1) chemical methods; (2) physical methods such asmicroinjection, electroporation, and particle bombardment; (3) viralvectors; (4) receptor-mediated mechanisms; and (5)Agrobacterium-mediated plant transformation methods. Further methods maybe used to accelerate DNA-coated metal particles into living cellsincluding, but not limited to, pneumatic devices; instruments utilizinga mechanical impulse or macroprojectile; centripetal, magnetic orelectrostatic forces; spray or vaccination guns; and apparatus based onacceleration by shock waves, such as electric discharge.

Further, in selecting the appropriate method for transforming cellsthere are additional variables or parameters that may be considered andtested, which are known to those skilled in the art. These may includephysical parameters such as: (1) the nature, chemical, and physicalproperties of the metal particles; (2) the nature, preparation, andbinding of the DNA onto the particles; and (3) the characteristics ofthe target plant tissue. These may also include environmental variablessuch as temperature, photoperiod and humidity of donor plants, explants,and bombarded tissues as well as biological factors.

In one exemplary embodiment, Agrobacterium-mediated transformation maybe used for transforming plants, more specifically crop plants such asmonocots and dicots exemplified by Nicotiana tabacum (tobacco), Brassicaspp. (canola), Solanum tuberosum (potato), Solanum lycopersicum(tomato), Zea mays (maize), Triticum spp. (wheat), Oryza sativa (rice),Papevar spp. (poppy), and xTriticosecale (triticale). There are severalAgrobacterium species that are known in the art, which can mediate thetransfer of the DNA, known as “T-DNA”. T-DNA may be geneticallyengineered to carry a specific piece of DNA of interest into selectedplant types or species. Some major events marking successfultransformation can include, but are not limited to, induction ofvirulence genes, processing and transfer of the T-DNA to the plant'sgenome.

Typically, prior to actual transformation, the nucleic acids or geneticcomponents of interest for introduction into plant cells or tissues areselected. Genetic components can include any nucleic acid that iscapable of being introduced into a plant cell or tissue. The geneticcomponents can include non-plant DNA, plant DNA, or synthetic DNA. In anexemplary embodiment, the genetic components of interest areincorporated into a DNA composition such as a recombinant,double-stranded DNA construct in the form of a plasmid or vectormolecule. DNA constructs in the form of plasmids or vectors typicallyconsist of a number of genetic components, including but not limited toregulatory elements such as promoters, leaders, introns, and terminatorsequences. The DNA construct may further comprise a number of geneticcomponents to facilitate transformation of the plant cell or tissue andregulate expression of the desired gene(s). Method for preparation ofDNA constructs in the form of plasmids or vectors containing the desiredgenetic components are well known in the art.

Promoters used in DNA constructs, which are active in plant cells areknown in the art. These promoters may include, but are not limited to,35S, 1′/2′, actin, tubulin, and chalcone synthase promoters. Suchpromoters can be used to create various types of DNA constructs forexpression in plants. Promoter hybrids can also be constructed toenhance transcriptional activity or to combine desired transcriptionalactivity, inducibility, and tissue or developmental specificity.

Genes or DNA of interest for use as a selectable, screenable, orscorable marker are exemplified by beta-glucuronidase (GUS), greenfluorescent protein (GFP), luciferase (LUC), antibiotics like kanamycinand hygromycin, and herbicides like glyphosate. Other selection devicescan also be implemented, including, but not limited to, tolerance tophosphinothricin, bialaphos, and positive selection mechanisms.

Any suitable plant transformation plasmid or vector can be used in thepresent invention with the methods disclosed herein. The plasmidconstruct may contain a selectable or screenable marker and associatedregulatory elements as described above, along with one or more nucleicacids, for example a structural gene or DNA of interest, expressed in amanner sufficient to confer a particular desirable trait selected plantcells. Examples of suitable structural genes may include, but are notlimited to, genes selected for modulating plant tolerance to insectand/or microbial pests, genes selected for modulating plant tolerance toherbicides, genes selected for conferring quality improvements to targetplant cells such as yield increases, nutritional enhancements, increasedtolerances to environmental and/or physiological stresses, or genessuitable for modulating any desirable changes in plant physiology,growth, development, morphology, or plant product(s).

One exemplary embodiment relates to a plant growth medium compositionfor modulation of plant transformation events. The composition containsa plant culture medium suitable for growing plants into which aneffective amount of at least one rare earth element-containing compoundis provided. Intermixing of the at least one rare earthelement-containing compound with the plant culture medium provides acomposition for increasing plant transformation frequency. A baselinelevel for plant transformation events is provided by culturing at leastone plant or plurality of plant cells on the plant culture medium whichdoes not contain the rare earth element-containing compound.

The rare earth element-containing compound may be selected from thegroup comprising CeCl₃, LaCl₃ and combinations thereof.

In a further embodiment, the plant growth media composition mayadditionally include compounds for further increasing the frequency ofplant transformation events. These compounds are exemplified bychloride-containing compounds, nitrate-containing compounds, andcombinations thereof.

The term “plant growth medium” as used herein, refers to the plantgrowth culture media, in any of liquid, solid, or semisolid form usedbefore, during, or after the transformation of the plant cells, tissues,parts, or other plant tissue explants and subsequent regeneration ofwhole, transgenic plants therefrom. Depending upon the plant speciesbeing transformed and the transformation process being used, the mediamay comprise one or more of isolation media, preculture media, inductionmedia, inoculation media, delay media, selection media, or regenerationmedia. The plant cells or tissues may include, but are not limited to,immature embryos, scutellar tissue, suspension cell cultures, immatureinflorescence, shoot apical meristem, nodal explants, callus tissue,hypocotyl tissue, cotyledons, roots, and leaves.

Another exemplary embodiment relates to a method for modulating thefrequency of plant transformation events. The method comprises the stepsof providing a plant culture medium composition suitable for growingplants comprising a plant culture medium and an effective amount of atleast one rare earth element-containing compound. Then, at least oneplant is contacted with the plant culture medium composition. Aplurality of plant cells from the plant are then transformed with anucleic acid of interest, after which, the plant cells are cultured andsubsequently assessed for the presence of transformation events. Ifdetected, the transformation events are quantified. The frequency of thequantified transformation events is then compared with a suitablecontrol. Changes in the quantified transformation events compared to thecontrol are indicative of a change in the frequency of planttransformation events.

Methods and Materials

Preparation of DNA Constructs:

DNA constructs were prepared using gene integration according tostandard molecular biology techniques, known to those skilled in theart. FIG. 1 illustrates exemplary structural arrangements of DNAconstructs containing the reporter markers LUC and hygromycin.

Agrobacterium tumefaciens strain GV3101, otherwise known as AtvirD2,(Tinland et al., EMBO J., 1995, 14(14): 3585-3595) carrying a selectablemarker encoding for a gene product for conferring resistance to theantibiotic rifampycin, was transformed with DNA construct comprising theLUC and hygromycin genes. The LUC and hygromycin genes were cloned inbetween two T-DNA borders, the left border (LB) and the right border(RB) allowing the processing by the Agrobacterium cells and delivery ofthe entire T-DNA portion. The Agrobacterium cells contained thescreenable, or scorable marker gene encoding for the LUC gene. The LUCmarker was used for quantifying transformation events. Hygromycinenabled selection of the transformants that were resistant to theantibiotic hygromycin.

Transformed Agrobacterium cells were selected by culturing thetransformed cells in a medium containing 50 μg/ml of spectinomycin. Thespectinomyin-resistant Agrobacterium cells were then harvested,re-plated onto fresh spectinomycin-containing media and the resultingcolonies were used to inoculate a 4-mL liquid culture containing YEBmedium supplemented with 10 mM magnesium sulfate, 100 μg/mL ofryphampycin, and 50 μg/mL of hygromycin.

The liquid culture was incubated overnight and 500 μL of theAgrobacterium culture was used to prepare a 100-mL culture.Agrobacterium cultures having optical densities in a selected range ofabout 1.5 to about 2.0 were collected, and washed with 10 mM ofmagnesium sulfate. A pellet was obtained by centrifugation after thewashing step, and was re-suspended in 50 mL of MS medium having a pH ofabout 5.2. This Agrobacterium suspension was vacuum infiltrated.

Detection of Homologous Recombination:

Homologous recombination was detected in plants, in particular tobaccoplants, that were transformed with scorable reporter markers, forexample, beta-glucuronidase (uidA or GUS). Upon homologousrecombination, the marker gene is restored. Homologous recombinationevents were identified using histochemical staining. An exemplaryimaging result is shown in FIG. 2, where the sites of homologousrecombination events on transformed plants are visualized as brightenedblue regions following histochemical staining and subsequent washingwith ethanol. A recombination substrate generally consists of twonon-functional overlapping copies of a GUS gene. Damage to one of theregions of homology may be repaired using the second copy as a template.A simple count of the number of recombination events in a population ofplants was used to conduct quantitative analyses of beta-glucuronidase(GUS) activity. The homologous recombination frequency (HRF) wasdetermined by relating the number of blue spots counted which areindicative of transformation events and then relating that number to thetotal number of plants scored. The recombination rate (RR) wasdetermined by relating the HRF to the total number of haploid genomespresent in the plant.

Counting of Regenerated Transformation Events:

The number of transgenic plants, having incorporated a marker gene,regenerated from tobacco calli in various transformation experimentswere counted. All the regenerated plants were screened using aluciferase camera. Spraying the transgenic plants with luciferine, thesubstrate for luciferase enzyme, allowed the identification oftransgenic plants expressing the luciferase.

Calculation of Genomic Number in Plants:

The total DNA of the transgenic lines was isolated from whole plants atpreferably the full rosette stage using a Nucleon™ PhytoPure™ plant DNAextraction kit from Amersham Life Science. DNA may also be isolated fromplants at a different development stage. The yield of total DNA measuredin one of micrograms per plant or micrograms per plant organ wascompared with the known mean DNA content, 0.16 pg of an A. thalianacell, to give an approximate number of genomes present in plants(Swoboda et al., Mol. Gen. Genet., 1993, 237(1-2): 33-40). The total DNAwas isolated from one of all leaves, roots, and stems of 4 plants pereach experimental group for each transgenic line. The average DNAcontent from these samples was used to estimate the number of genomespresent.

For calculation of the approximate number of genomes present in lateraland medial parts of the leaves, the leaves were cut into two halves.Twelve groups of 8 leaves each were prepared and DNA content measured.The total amount of DNA measured from the lateral or medial part of the8 leaves was divided by number of leaves used to get an average DNAcontent per leaf. The DNA content was also measured for nine groups ofplants sampled at the age of 2, 3, 5, 7, 10, 13, 16, 19 and 22 dayspost-germination, where between about 4-60 plants were present in eachgroup.

To determine whether the DNA extraction method had a significantinfluence on the DNA yield, the DNA was isolated and content measuredusing an alternate protocol. The tissue from four 3 week-old Arabidopsisplants were snap frozen, grinded, and homogenized in 400 uL of anextraction buffer (200 mM Tris-Cl pH 5; 250 mM NaCl; 25 mM EDTA; 0.5%SDS), and transferred to 1.5 mL Eppendorff tubes. After the addition of6 uL of 2-mercaptoethanol, the tubes were vortexed and stored at about65° C. for a period of 30 to 45 minutes with occasional vortexing. Thetubes were then centrifuged for a period of about 5 minutes at 3300 rpm,after which the supernatant was collected and transferred to new tubes.An equal volume of phenol was added to each of the tubes and the tubeswere then mixed vigorously for a period of about 20 to 30 seconds. Aftercentrifugation at a maximum speed 12,000 rpm for a period of about 2minutes, the aqueous upper phase material was then collected andtransferred to new tubes. An equal volume of chloroform was added toeach of the tubes and the contents were well mixed. Tubes were thencentrifuged at a maximum speed of 12,000 rpm for a period of about 2minutes. The upper aqueous phase material was again transferred to newtubes and RNAase was added to a final concentration of 20 ug/mL. Thetubes were then incubated for about 30 minutes at 37° C., and a 1/10volume of 3M sodium acetate, pH 5.0 and 1 volume of cold isopropanolwere added to each tube. The tubes were stored for about 30 minutes at−20° C. and then centrifuged at a maximum speed of 12,000 rpm for about15 minutes. Pellets of material collected from the tubes were washedwith 1 mL of cold, 70% ethanol, centrifuged at a maximum speed of 12,000rpm for a period of about 5 minutes, and then dried and re-suspended insterile distilled de-ionized water. DNA contents were then measured on aspectrophotometer.

While the DNA yields were somewhat different between the two methodsused, the ratio between the amounts of DNA in plants grown at differentconditions was the same. For the experiments detailed below, theNucleon™ PhytoPure™ plant DNA extraction method was used.

Bacterial Culture:

The Agrobacterium cultures were streaked on plates containing solid YEPmedium supplemented with a suitable antibiotic, for example hygromycin.The plates were incubated at 28° C. overnight. A single colony was thenused to start a small 3 ml liquid culture of YEP supplemented withantibiotics. The 3-ml bacteria culture was incubated overnight at 28° C.in a rotary incubator between about 190-200 rpm. The 3-ml liquid culturewas used to inoculate a primary 150 ml culture that was then grownovernight under the same conditions. Following incubation, bacteria wereharvested (5000 rpm, 5 min) and re-suspended in ½-strength MS medium toa final optical density of 0.6 measured at 600 nm. The resultantbacterial suspension was then supplemented with a 100 mM acetosyringonesolution to a final concentration of 100 uM. The bacterial suspensionwas then incubated for at least 30 minutes to stimulate the bacteria.Following incubation with acetosyringone, the bacteria were used fortransformation.

Plant Growth Conditions:

Seeds of tobacco cultivar Big Havana were surface-sterilized with asolution containing 1% bleach and 0.05% Tween-80 for about 3 minutes andthen rinsed twice with sterile distilled water for about 5 minutes.Surface-sterilized seeds were plated in 100 mm Petri dishes on sterileWhatman® filter paper submersed in 4 ml of liquid MS medium containingvarying amounts of CeCl₃, LaCl₃ and a combination of CeCl₃ and LaCl₃ andthe plants were transferred to a growth chamber for germination. Oncegerminated, the plants were removed from the growth chamber and grownfor a period of one week under conditions of 16-hours light, 22° C. and8-hours dark, 18° C. The three to five one week old plants were thenremoved from the 4-ml liquid medium and transferred to single sterileglass 250 ml flasks containing 15 ml of sterile liquid MS mediasupplemented with varying amounts of CeCl₃, LaCl₃ and a combination ofCeCl₃ and LaCl₃. Flasks were then installed on shakers at 50-75 rpm.Plants were continuously grown under conditions of 16-hours light, 22°C. and 8-hours dark, 18° C. The growth medium in each flask was replacedweekly with 25 ml of fresh medium. Following a 3-week period, plantswere removed from the flasks and 2 to 3 pairs of fully developed freshleaves about 2-4 cm long were harvested (cut from the plant) fortransformation with Agrobacteria.

Plant Growth Media:

Murashige Skoog (“MS”) medium was used as the base plant growth medium.Standard MS medium generally contains 20.6 mM of ammonium nitrate and18.8 mM of potassium chloride. Other plant growth media known to thoseskilled in the art may also be used, such as the Gamborg's B5 medium orChu's N6 medium.

Plant Transformation:

Experimental groups of tobacco plants were germinated and grown in aliquid medium culture supplemented with varying amounts of CeCl₃, LaCl₃and a combination of CeCl₃ and LaCl₃. Control plants for thetransformation experiments were grown in a standard MS-medium that wasnot supplemented with CeCl₃ or LaCl₃.

Four weeks post-germination, the tobacco plants were removed from theliquid medium culture. The leaves from the plants were removed, andseveral parallel incisions were made along the leaves. The leaves werethen vacuum infiltrated with an Agrobacterium suspension culturecarrying the plasmid with LUC (gene coding for luciferase) andhygromycin (gene coding for the resistance to hygromycin) genes.

The leaves were vacuum-infiltrated twice for about 5 minutes with theAgrobacterium suspension culture using standard procedures known in theart. Following vacuum infiltration, the tobacco leaves were dried forabout 5 to 10 minutes on sterile Whatman® filter paper to removesubstantially all excess Agrobacterium cells. The leaves were thenplaced on plates containing MS medium, and each of the plates wereplaced for in a room for a period of 3 day at a temperature of 22° C.and exposed to a daily regime of 16-hours of light and 8-hours of dark.

Leaves from each of the experimental groups grown on the different mediacompositions having varying amounts of CeCl₃, LaCl₃ and a combination ofCeCl₃ and LaCl₃ and the controls were washed with sterile water toremove the Agrobacterium suspension. To remove traces of growth medium,leaves were blotted on sterile filter paper and then submersed in aPetri dish laid out with Whatman® filter paper containing re-suspendedAgrobacterium cells. Each submersed leaf surface was incised using asharp surgical blade in parallel along the side veins. The distancebetween the two parallel incisions was about 5-7 mm. The primary leafvein and leaf margins were left intact. Once cutting was complete,leaves remained submersed for a period of about 10 minutes. Leaves werethen removed from the Petri dish and were blotted dry and placedupside-down on plates of MS medium, and were incubated in a dark room at22° C. for a period of 3 days. Following incubation, leaves were removedfrom the plates and rinsed with sterile distilled water, blotted dry andtransferred onto solid MS medium containing 0.8 mg/L of indole-3-aceticacid (IAA), and 2 mg/L kinetin for calli induction and regeneration, acombination of 100 mg/L ticarcillin and 3 mg/L potassium clavulanate tocontrol Agrobacterium growth, and 25 mg/L hygromycin for selection fortransformed cells.

After a period of about 3 to 4 weeks, the number of regenerated calliwere determined. Shoots that developed were excised from calli andtransferred to a root inducing solid MS medium containing 0.5 mg/L ofnaphtaleneacetic acid (NAA), 100 mg/L ticarcillin, 3 mg/L potassiumclavulanate and 25 mg/L hygromycin. After a 1 to 2 week period of rootinduction, the plantlets were transplanted to soil.

EXAMPLES Example 1 Effect of CeCl₃ and LaCl₃ Compounds on Plant Growth

In the selection of rare earth element-containing compounds forsupplementing plant growth medium, the effects of both CeCl₃ and LaCl₃on plant growth were evaluated.

It was identified that a concentration of less than 3 μM does not resultin physiological damage to plants.

Example 2 Analysis of Homologous Recombination in Nicotiana tabacum (N.tabacum)

The effects of rare earth elements, specifically CeCl₃ and LaCl₃, onplant transformation were measured using transgenic plants germinatedfrom N. tabacum line #LU2 produced by Dr. Igor Kovalchuk, University ofLethbridge, Alberta. Plants were germinated and grown on a solid ormodified MS basal medium in presence of varying quantities of CeCl₃ andLaCl₃.

The frequency of homologous recombination was measured in approximately100 N. tabacum plants in each experimental group, germinated and grownon a solid control medium or on a modified solid media containing one of0.1 μM CeCl₃, 0.1 μM LaCl_(3,) 0.1 μM of each CeCl₃ and LaCl₃, 0.3 μMCeCl₃, 0.3 μM LaCl_(3,) 0.3 μM of each CeCl₃ and LaCl₃, 1.0 μM CeCl₃,1.0 μM LaCl_(3,) 1.0 μM of each CeCl₃ and LaCl₃, 2.0 μM CeCl₃, 2.0 μMLaCl_(3,) 2.0 μM of each CeCl₃ and LaCl₃, 3.0 μM CeCl₃, 3.0 μM LaCl_(3,)and 3.0 μM of each CeCl₃ and LaCl₃, for a period of about 4 weeks. Theexperiments were performed in triplicate.

The media compositions used to determine the effects of the rare earthelements on recombination frequency and transformation efficiency in N.tabacum are listed below in Table 1.

TABLE 1 Experimental media compositions, all final concentrations(except CeCl3 and LaCl3, that are in μM) listed in mM Ce/ Ce/ MS mediaCe La Ce/La Ce La Ce/La Ce La Ce/La La La (mM) Ct 0.1 0.1 0.1 0.3 0.30.3 1.0 1.0 1.0 Ce 2.0 La 2.0 2.0 Ce 3.0 La 3.0 3.0 NH₄NO₃ 20.6 39.439.4 39.4 39.4 39.4 39.4 39.4 39.4 39.4 39.4 39.4 39.4 39.4 39.4 39.439.4 KNO₃ 18.8 18.8 18.8 18.8 18.8 18.8 18.8 18.8 18.8 18.8 18.8 18.818.8 18.8 18.8 18.8 18.8 CaCl₂ 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 MgSO₄1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5KH₂PO₄ 1.25 1.25 1.25 1.25 1.25 1.25 1.25 1.25 1.25 1.25 1.25 1.25 1.251.25 1.25 1.25 1.25 CeCl3 0.1 0.1 0.3 0.3 1.0 1.0 2.0 2.0 3.0 3.0 LaCl30.1 0.1 0.3 0.3 1.0 1.0 2.0 2.0 3.0 3.0

The homologous recombination frequency was measured using histochemicalstaining for each of the plants grown on the control and modified mediumcompositions as shown in FIG. 3. N. tabacum plants that were grown onthe modified growth media having between about 0.1 μM-2.0 μM CeCl₃, andbetween about 0.3 μM-1.0 μM of each CeCl₃ and LaCl₃, resulted in atleast a 1-fold increase in homologous recombination when compared toplants grown on control MS medium. N. tabacum plants that were grown onthe modified growth media having one of 1.0 μM CeCl₃, 1.0 μM LaCl_(3,)and 1.0 μM of each CeCl₃ and LaCl₃ resulted in at least a 2-foldincrease in homologous recombination when compared to plants grown oncontrol MS medium. Further, modified growth media having 1.0 μM of eachCeCl₃ and LaCl₃, exhibited a 4.0-fold increase in homologousrecombination when compared to the plants grown on the control MS medium(Student's t-test, α=0.05). The results of these experimentsdemonstrated that presence of CeCl₃ and LaCl₃ in a growth medium,particularly at an optimized concentration, significantly increased thefrequency of homologous recombination.

Example 3 Analysis of the Effects of CeCl₃ on Calli Regeneration in N.tabacum

The effects of CeCl₃ on the occurrence calli regeneration in N. tabacumplants were evaluated. Calli were regenerated under selective conditionsutilizing a selection marker of hygromycin, 25 mg/L.

N. tabacum plants were grown on liquid MS media supplemented with 0.1and 0.5 μM of CeCl₃ as shown in Table 2. Plants grown in presence of 0.1and 0.5 μM of CeCl₃ in liquid media were used for transformation withluciferase containing a T-DNA construct. Calli were regenerated underselective conditions (hygromycin, 25 mg/L).

TABLE 2 Integration frequency Calli regenerated and transplanted LUC LUCexpression test positive leaves LUC LUC died plants/leaf CeCl₃transformed positive negative total on soil transformed   0 μM 20 15 419 3 0.75 0.1 μM 20 23 13 35 7 1.15 0.5 μM 20 38 18 56 8 1.9

Media containing 0.1 and 0.5 μM of CeCl₃ increased the number of calliregenerated by factor of 1.8-fold and 2.9-fold respectively whencompared to the control medium as shown in Table 2.

A second independent experiment was performed and N. tabacum plants weregrown on liquid MS media supplemented with 0.1 and 1.0 μM of CeCl₃ asshown in Table 3. Plants grown in presence of 0.1 and 1.0 μM of CeCl₃ inliquid medium were used for transformation with luciferase containing aT-DNA construct. Calli were regenerated under selective conditions(hygromycin, 25 mg/L).

TABLE 3 Integration frequency Calli regenerated and transplanted LUC LUCexpression test positive leaves LUC LUC died plants/leaf CeCl₃transformed positive negative total on soil transformed   0 μM 20 17 522 4 0.85 0.1 μM 20 34 13 48 6 1.7 1.0 μM 20 57 18 73 11 2.85

Media containing 0.1 and 1.0 μM of CeCl₃ increased the number of calliregenerated by factor of 2.2-fold and 3.3-fold respectively whencompared to the control medium as shown in FIG. 4.

Example 4 Analysis of the Effects of CeCl₃ on the Frequency of StableT-DNA Integrations in N. tabacum

The effects of CeCl₃ on the occurrence of stable plant transformationevents in N. tabacum plants were evaluated. These experiments identifiedtransformed plants where the DNA of interest had stably integrated intothe plant genome.

N. tabacum plants were grown on liquid MS media supplemented with 0.1and 1.0 μM of CeCl₃ as shown in Table 3. The newly appeared plantletsregenerated from the calli of Example 3, showing evidence of rootformation, were excised from the calli and transferred to soil.

Plantlets were sprayed with luciferine, and the total number ofluciferase-positive plantlets was scored. This allowed the calculationof the transformation frequency, as the number of plants expressing LUCgene to the number of transformed leaves, shown in FIG. 4.

The number of stable transformants re-generated from plants grown onCeCl3 supplemented media and control medium, as detailed in Example 3,were compared. The comparison of the numbers of stable transformantsformed on the media containing 0.1 and 1.0 μM of CeCl₃ and the controlmedia showed a 2.0- and 3.4-fold difference, respectively as shown inFIG. 4.

The above-described embodiments have been provided as examples, forclarity in understanding the invention. A person of skill in the artwill recognize that alterations, modifications and variations may beeffected to the embodiments described above while remaining within thescope of the invention as defined by the claims appended hereto.

1. A plant culture medium composition for modulating planttransformation events, the composition comprising: a plant culturemedium; and an effective amount of at least one compound having arare-element component intermixed thereinto.
 2. The compositionaccording to claim 1, wherein said composition further includesadditional compounds for increasing the frequency of planttransformation events.
 3. The composition according to claim 1, whereina baseline level for plant transformation events is provided byculturing at least one of a plurality of plant cells, at least oneplant, and plant tissue on said plant culture medium.
 4. The compositionaccording to claim 1, wherein a baseline level for plant transformationevents is provided by culturing at least one plant on said plant culturemedium.
 5. The composition according to claim 1, wherein said at leastone rare-element-containing compound is selected from the groupcomprising CeCl₃, LaCl₃ and combinations thereof.
 6. The compositionaccording to claim 1, wherein said at least one rare earthelement-containing compound is CeCl₃.
 7. The composition according toclaim 1, wherein said at least one rare earth element-containingcompound is LaCl₃.
 8. The composition according to claim 1, wherein saidat least one rare earth element-containing compound is a combination ofat least CeCl₃ and LaCl₃.
 9. The composition according to claim 6,wherein said CeCl₃ is present in an amount greater than about 0.10 μMand less than about 3.0 μM.
 10. The composition according to claim 7,wherein said LaCl₃ is present in an amount greater than about 0.10 μMand less than about 3.0 μM.
 11. The composition according to claim 8,wherein each of said CeCl₃ and LaCl₃ is present in an amount greaterthan about 0.10 μM and less than about 3.0 μM.
 12. The compositionaccording to claim 2, wherein on comparison to said baseline level forplant transformation events, said composition increases the number ofplant transformation events.
 13. The composition according to claim 2,wherein on comparison to said baseline level for plant transformationevents, said composition increases the number of plant transformationevents by at least one of 2-fold, 3-fold, 4-fold, 5-fold, and 10-fold.14. The composition according to claim 2, wherein said plurality ofplant cells is selected from the group comprising immature embryos,scutellar tissue, suspension cell cultures, immature inflorescence,shoot apical meristem, nodal explants, callus tissue, hypocotyl tissue,cotyledons, roots, and leaves.
 15. The composition according to claim 1,wherein a plant cell grown on said plant culture medium composition isselected from the group comprising immature embryos, scutellar tissue,suspension cell cultures, immature inflorescence, shoot apical meristem,nodal explants, callus tissue, hypocotyl tissue, cotyledons, roots, andleaves.
 16. The composition according to claim 1, wherein a plant grownon said plant culture medium composition is selected from the groupcomprising monocots and dicots.
 17. The composition according to claim1, wherein a plant grown on said plant culture medium composition isselected from the group comprising Arabidopsis sp., Nicotiana tabacum,Brassica spp., Solanum lycopersicum, Solanum tuberosum, Zea mays,Triticum spp., Oryza sativa, Papevar spp. and x Triticosecale.
 18. Thecomposition according to claim 2, wherein said plant tissue is isolatedfrom a plant grown on said plant culture medium composition is selectedfrom the group comprising Arabidopsis sp., Nicotiana tabacum, Brassicaspp., Solanum lycopersicum, Solanum tuberosum, Zea mays, Triticum spp.,Oryza sativa, Papevar spp. and x Triticosecale.
 19. The compositionaccording to claim 1, wherein said plant culture medium is selectingfrom the group comprising isolation media, pre-culture media, inductionmedia, inoculation media, delay media, selection media, and regenerationmedia.
 20. A method for modulating the frequency of plant transformationevents, the method comprising the steps of: a) providing the plantculture medium composition of claim 1; b) contacting at least one plantwith the plant culture medium composition; c) transforming at least onecell from said at least one plant with a nucleic acid of interest; d)detecting the presence of at least one transformation event, and e)quantifying said transformation event; f) comparing the frequency ofsaid quantified transformation events with a suitable control; whereinchanges in the quantified transformations events compared to the controlare indicative of a change in the frequency of plant transformationevents.
 21. The method according to claim 20, wherein said compositionfurther includes additional compounds for increasing the frequency ofplant transformation events.
 22. The method according to claim 20,wherein said at least one rare earth element-containing compound isselected from the group comprising CeCl₃, LaCl₃ and combinationsthereof.
 23. The method according to claim 20, wherein said at least onerare earth element-containing compound is CeCl₃.
 24. The methodaccording to claim 20, wherein said at least one rare earthelement-containing compound is LaCl₃.
 25. The method according to claim20, wherein said at least one rare earth element-containing compound isa combination of at least CeCl₃ and LaCl_(3.)
 26. The method accordingto claim 23, wherein said CeCl₃ is present in an amount greater thanabout 0.10 μM and less than about 3.0 μM.
 27. The method according toclaim 24, wherein said LaCl₃ is present in an amount greater than about0.10 μM and less than about 3.0 μM.
 28. The method according to claim25, wherein each of said CeCl₃ and LaCl₃ is present in an amount greaterthan about 0.10 μM and less than about 3.0 μM.
 29. The method accordingto claim 20, wherein said suitable control is selected from the groupcomprising a stored dataset of results generated from studies of thepresence and expression transformation events in one or morepopulation(s) of plants grown on said plant culture medium, a storeddataset of results generated from studies of the presence and expressionof transformation events in one or more population(s) of plant cellsgrown on said plant culture medium, a stored dataset of resultsgenerated from studies of the presence and expression transformationevents in one or more population(s) of plant tissue grown on said plantculture medium and combinations thereof.
 30. The method according toclaim 20, wherein said suitable control is a baseline level for planttransformation events and is provided by culturing at least one of aplurality of plant cells, at least one plant, and plant tissue on saidplant culture medium.
 31. The method according to claim 20, wherein saidchanges in the quantified transformations compared to the control, arean increase in the frequency of plant transformation events.
 32. Themethod according to claim 20, wherein said changes in the quantifiedtransformations compared to the control are an increase in the frequencyof plant transformation events by at least one of 2-fold, 3-fold,4-fold, 5-fold, and 10-fold.
 33. The method according to claim 20,wherein said at least one plant is selected from the group comprisingmonocots and dicots.
 34. The method according to claim 20, wherein saidat least one plant is selected from the group comprising Arabidopsissp., Nicotiana tabacum, Brassica spp., Solanum lycopersicum, Solanumtuberosum, Zea mays, Triticum spp., Oryza sativa, Papevar spp. and xTriticosecale.
 35. The method according to claim 20, wherein said plantculture medium is selecting from the group comprising isolation media,pre-culture media, induction media, inoculation media, delay media,selection media, and regeneration media.
 36. A method for transforming aplant cell, the method comprising the steps of: a) providing the plantculture medium composition of claim 1; b) contacting a plurality ofplant cells with the plant culture medium composition; c) transformingsaid plurality of plant cells with a nucleic acid of interest; d)detecting the presence of at least one transformation event; and e)regenerating at least one transformed plant from at least onetransformed plant cell.
 37. The method according to claim 36, whereinsaid transformed plant cells produces a transgenic plant.
 38. The methodaccording to claim 36, wherein said composition further includesadditional compounds for increasing the frequency of planttransformation events.
 39. The method according to claim 36, whereinsaid at least one rare earth element-containing compound is selectedfrom the group comprising: CeCl₃, LaCl₃ and combinations thereof. 40.The method according to claim 36, wherein said at least one rare earthelement-containing compound is CeCl₃.
 41. The method according to claim36, wherein said at least one rare earth element-containing compound isLaCl₃.
 42. The method according to claim 36, wherein said at least onerare earth element-containing compound is a combination of at leastCeCl₃ and LaCl₃.
 43. The method according to claim 40, wherein saidCeCl₃ is present in an amount greater than about 0.10 μM and less thanabout 3.0 μM.
 44. The method according to claim 41, wherein said LaCl₃is present in an amount greater than about 0.10 μM and less than about3.0 μM.
 45. The method according to claim 42, wherein each of said CeCl₃and LaCl₃ is present in an amount greater than about 0.10 μM and lessthan about 3.0 μM.
 46. The method according to claim 36, wherein saidplurality of plant cells is selected from the group comprising immatureembryos, scutellar tissue, suspension cell cultures, immatureinflorescence, shoot apical meristem, nodal explants, callus tissue,hypocotyl tissue, cotyledons, roots, and leaves.
 47. The methodaccording to claim 36, wherein said plant culture media is selectingfrom the group comprising isolation media, pre-culture media, inductionmedia, inoculation media, delay media, selection media, and regenerationmedia.
 48. Use of an effective amount of a rare earth element-containingcompound intermixed into a plant culture medium to modulate planttransformation.
 49. The use according to claim 48, wherein said rareearth element-containing compound is selected from the group comprisingCeCl₃, LaCl₃ and combinations thereof.
 50. The use according to claim48, wherein said rare earth element-containing compound is CeCl₃. 51.The use according to claim 48, wherein said rare earthelement-containing compound is LaCl₃.
 52. The use according to claim 48,wherein said rare earth element-containing compound is a combination ofat least CeCl₃ and LaCl₃.
 53. The use according to claim 50, whereinsaid CeCl₃ is present in an amount greater than about 0.10 μM and lessthan about 3.0 μM.
 54. The use according to claim 51, said LaCl₃ ispresent in an amount greater than about 0.10 μM and less than about 3.0μM.
 55. The use according to claim 52, each of said CeCl₃ and LaCl₃ ispresent in an amount greater than about 0.10 μM and less than about 3.0μM.
 56. The use according to claim 48, wherein said plant culture mediumis selecting from the group comprising isolation media, pre-culturemedia, induction media, inoculation media, delay media, selection media,and regeneration media.